Colonization dynamic and distribution of the endophytic fungus Microdochium bolleyi in plants measured by qPCR

Microdochium bolleyi is a fungal endophyte of cereals and grasses proposed as an ideal model organism for studying plant-endophyte interactions. A qPCR-based diagnostic assay was developed to detect M. bolleyi in wheat and Brachypodium distachyon tissues using the species-specific primers MbqITS derived from the ITS of the ribosomal gene. Specificity was tested against 20 fungal organisms associated with barley and wheat. Colonization dynamics, endophyte distribution in the plant, and potential of the seed transmission were analyzed in the wheat and model plant B. distachyon. The colonization of plants by endophyte starts from the germinating seed, where the seed coats are first strongly colonized, then the endophyte spreads to the adjacent parts, crown, roots near the crown, and basal parts of the stem. While in the lower distal parts of roots, the concentration of M. bolleyi DNA did not change significantly in successive samplings (30, 60, 90, 120, and 150 days after inoculation), there was a significant increase over time in the roots 1 cm under crown, crowns and stem bases. The endophyte reaches the higher parts of the base (2–4 cm above the crown) 90 days after sowing in wheat and 150 days in B. distachyon. The endophyte does not reach both host species’ leaves, peduncles, and ears. Regarding the potential for seed transmission, endophyte was not detected in harvested grains of plants with heavily colonized roots. Plants grown from seeds derived from parental plants heavily colonized by endophyte did not exhibit any presence of the endophyte, so transmission by seeds was not confirmed. The course of colonization dynamics and distribution in the plant was similar for both hosts tested, with two differences: the base of the wheat stem was colonized earlier, but B. distachyon was occupied more intensively and abundantly than wheat. Thus, the designed species-specific primers could detect and quantify the endophyte in planta.


Introduction
Endophyte communities are found in almost all plants [1,2].These communities usually consist mainly of bacteria or fungi [3] that reside inside plant organs and do not damage the host tissue [3][4][5][6][7].Some fungal endophytes can spend part of their life cycle as latent pathogens in plant tissues [8][9][10].Fungal endophytes are vital in enhancing plant resilience against various stresses, such as abiotic factors like drought and biotic factors like pathogens and pests [11][12][13].
The first objective of this study was to develop species-specific primers for qPCR detection and quantification of endophyte M. bolleyi in plant tissues and thorough testing of their specificity and capability of detecting the endophyte in plants.The second aim of the study was to assess the colonization dynamic over time, the location of the endophyte in plants, and the seed transmission potential of the endophytic fungus M. bolleyi using wheat and the model plant species B. distachyon.

Biological materials
All plants were cultivated in a cooled greenhouse (20/18˚C, day/night) within pots 8 cm in diameter filled with a 50:50 planting substrate-sand (vol:vol) mixture, the substrate FLOR-COM SV (BB Com, Letohrad, Czech Republic).For the experiment were used not treated seeds of the wheat cultivar Bohemia and of B. distachyon line Bd21 obtained from the Joint Genome Institute (https://jgi.doe.gov).Ten seeds were placed into each pot.After germination, the number of plants in every pot was reduced to six.
Six Mb isolates were used (UPOC-FUN-253-258) from the Collection of Phytopathogenic Microorganisms UPOC (Czech Republic).Inoculum of Mb isolates was obtained by cultivation on double sterilized millet seed [32], which was previously tested for the absence of Mb by qPCR.The inoculation of the plants was also carried out according to the method of Matus ˇinsky et al. (2022) by adding inoculum to the seeds during the sowing [32].Only sterile millet without endophyte was added to the control treatments.
Isolates of other fungi used for testing primers were obtained from four different collections of microorganisms (Table 1).

Sampling and assessment
To evaluate endophyte colonization by qPCR, plants were collected 30, 60, 90, 120, and 150 days after sowing/inoculation (dai), surface sterilized for 3 min in 1% NaOCl, and thoroughly rinsed in sterilized distilled water to remove all superficial hyphae and then dried.Plants were cut into individual parts: the lower part of the roots (distal part of the roots), roots 1 cm (part of the roots 1 cm below the crown), crown, base 1 cm (part of the stem base 1 cm above the crown), base 2-4 cm (part of the stem base 2-4 cm above the crown), peduncles, and ears (S1 Fig) .To evaluate endophyte colonization by light microscopy, plants were collected in the same terms as above (30,60,90,120, and 150 dai), and then fixed in 70% ethanol.Plants fixed in 70% ethanol were cleared in 2.5% KOH for three days, acidified in 1% HCl and stored in lactoglycerol [14].Colonization of plant tissues by the endophytic fungus was assessed by microscopic examination (200× magnifications).Results were evaluated as positive when the visible presence of Mb chlamydospores was observed (S2 Fig) .Estimation of root colonization level was made by an adapted method according to Trouvelot et al. (1986) [40].
Seeds of massively colonized plants were harvested and subsequently tested for Mb DNA by qPCR and sown to analyze the potential for endophyte transfer by seed.Plants grown from

DNA isolation, primers design, and qPCR
Fungal mycelia (approximately 50-100 mg of biomass) of tested fungi (Table 1) were harvested from Petri dishes, ground to a fine powder in a cooled mortar using liquid nitrogen, then homogenized.Total genomic DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Germany).Plant material (approximately 50 mg of dried biomass per sample) was also ground to powder and extracted as described above.DNA concentration was measured using Qubit fluorometric quantification (ThermoFisher Scientific, Waltham, MA, USA), and DNA was diluted to a concentration of 5 ng μL −1 .
Based upon sequences ITS (KP859018), LSU (large subunit of the ribosomal gene; KP858954), and RPB2 gene (RNA 40 polymerase II second-largest subunit (KP859127)) [41] selected from the GenBank database, five primer pairs (MbqITSF/R, MbqLSU1F/R, MbqLSU2F/R, MbqPOL1F/R, and MbqPOL2F/R) were designed using Primer3Plus software [42].The samples were analyzed using the CFX96TM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA).The quantitative PCR mix consisted of 1× SYBR Green (Top-Bio, Vestec, Czech Republic), 0.2 μM forward and reverse primers (the best final primers MbqITSF/R were selected from a preliminary screen of the designed primers; (S1 Table )), 10 ng DNA (2 μL), and water to final volume 15 μL.The reference gene for wheat was TaPAL [43], and for Bd, it was BdFIM [44].The control samples consisted of DNA of wheat or Bd plants colonized by the known level of root colonization by Mb obtained from the previous experiment; it will be further described in this article as a standard sample (sample of roots 1 cm under crown collected 90 days after sowing; wheat 20.6% and Bd with 23.4% of colonization measured by light microscopy method according to Trouvelot et al. (1986) [40]).The primers' specificity and presence of primer dimers were verified by melting analysis.For the resulting MbqITSF/R primers, the reaction efficiency was analyzed by qPCR with DNA of Mb (isolate UPOC-FUN-253) and dilution series (1.0, 0.1, 0.01, 0.001, and 0.0001 ng).

Test of primers specificity and diagnostic potential in plant tissues
The primers were tested on the six Mb isolates as described above.The designed primer pairs were tested for their specificity towards the DNA of fungi associated with diseases of wheat and other cereals.Fungal cultures were obtained from four microorganism collections (Table 1) and cultured on PDA.Furthermore, the potential for detection was tested in wheat, and Bd inoculated with the Mb compared with non-inoculated plants (inoculation and sampling as described above).All reactions were repeated three times.

Statistical analysis
The data were analyzed using the 2 -ΔΔCq method with CFX Maestro software (Bio-Rad), and three biological and three technical replicates were run.The reaction efficiency was calculated using CFX Maestro software (Bio-Rad) from the slope value of the standard curve according to the formula E = (-1 + (10-1/slope) × 100) [45].Differences in the relative quantity of the average values of inoculated samples were evaluated and compared to the standard sample (described above) by Tukey's pairwise comparison test (P < 0.05; CFX Maestro software, Bio-Rad).

Demonstrated root colonization by Mb of wheat and Bd
Using microscopic and molecular methods, the presence of Mb was detected in both wheat and Bd samples.Using light microscopy, chlamydospores were observed in the seed coats, roots, crowns, and stem bases of wheat and Bd (S2, S3 Tables and Fig 1).No presence of chlamydospores was observed in plants without inoculation.
A set of MbqITSF/R best met the specificity requirements of the five designed primer pairs.The primers showed positive results in all six tested isolates of Mb (S4 Table).In the specificity test, the best-selected primers amplified DNA only from Mb, not any other fungal pathogens screened (Table 1).The primer set was therefore used to examine Mb-inoculated and noninoculated plant material (wheat and Bd).Inoculated plants with chlamydospores present in plant tissues of both wheat and Bd showed positive results after qPCR with primers MbqITSF/ R and, on the contrary, non-inoculated plants without chlamydospores in plant tissues showed negative results with Cq values higher than 30.The analysis confirmed that the primers were specific and robust in detecting and quantifying Mb within plant tissues.No false positive results were seen after melting analysis.Other tested primer pairs, MbqLSU1F/R, MbqLSU2F/ R, MbqPOL1F/R, and MbqPOL2F/R, failed in the specificity test and were excluded from other parts of this study.The efficiency of the reaction was determined using a dilution series of DNA from the Mb isolate (UPOC-FUN-253).Based on the standard curve obtained, the efficiency (E) of the reaction was found to be 98.5% (S5 Table ).The method showed a reliable positive response at 0.001 ng of Mb DNA (S5 Table ), and the melting curve gives a single peak, indicating no artifacts.

Dynamics of Mb colonization in wheat and Bd over time
The seed coats, i.e., the already dead tissue, are massively colonized (30 days after inoculation) (Fig 1C and 1D).Only then are the living parts of the plant inhabited, such as the crown and roots, especially the part of the roots near the crown (30 dai).Over time (90 dai), the endophyte spreads to the distal parts of the roots and in wheat to the higher parts of the stem, 2-4 cm, but in Bd, only up to 1 cm and not higher.The DNA of the endophyte increases over time in the aerial parts, in wheat earlier than in Bd (Fig 2).In the underground parts, especially in the lower distal part of the roots, however, the level of endophyte DNA is growing very slowly during sampling periods (30-150 dai) with no significant changes (Fig 2).In the wheat aboveground parts, the presence of chlamydospores is confirmed microscopically in the outer sheath of the oldest leaf sheaths enclosing the stem (60-90 dai).Gradually, chlamydospores can be found in the sheaths of the second-oldest and third-oldest leaves (120-150 dai).Usually, the sheaths of leaves already in the senescence stage are colonized in this way, so a tendency for Mb to inhabit sheaths already in the senescence stage can be observed.On microscopy, it was found that most of the chlamydospores in the aerial part of the plants are in the cells adjacent to the vascular bundles (Fig 1A and 1B).Still, the fungus does not penetrate the vascular bundle itself, and the tissues are free of visual symptoms, i.e., without browning or other signs of damage.At the last sampling date of 150 days after sowing, the plants were already completely dry and dead, and the increase in Mb DNA in crown and roots 1cm above crown in wheat and stem base 2-4 cm above crow in Bd is, therefore, more likely due to the saprotrophic effect of the fungus.No Mb DNA was detected in leaf blades, peduncles, ears, and harvested seeds, nor was the presence of chlamydospores seen in these parts of the plants (S2 and S3 Tables).

Distribution of Mb in wheat and Bd plants
The endophyte distribution in the plant varies over time, as indicated in the previous section of the results.Endophyte colonizes the roots, crowns, and stem bases of plants.The parts of the root adjacent to the crown are more intensively colonized than the distal parts of the roots, which are less colonized (S2 and S3 Tables).Crowns are inhabited initially, and the intensity is higher in the last phase of the plant's life.The first tissues that are colonized after sowing are the seed coats (Fig 1C and 1D).Surprisingly, the plant's above-ground parts, especially the lowest parts of the base, are inhabited significantly, and the intensity increases over time.The higher parts of the base, 2-4 cm, are also colonized in wheat.In contrast, in Bd, colonization in these higher regions of the base occurs only at the last sampling date, 150 days after sowing/ inoculation.Neither leaf blades nor peduncles and ears are colonized even at the late stages of plant development (S2 and S3 Tables).

Transmission by seed was not confirmed
Seeds from ten plants heavily colonized by the endophyte Mb were harvested and sown.These seeds and the plants grown from these seeds were screened for the presence of Mb by both light microscopy and qPCR.Neither the seeds nor any parts of the plants sown from these seeds contained endophyte Mb (S6 Table ).Thus, it can be construed that Mb is not transmitted vertically, i.e., by seed.

Discussion
The fungal endophyte of grasses and cereals Mb is a suitable model organism for studying endophyte interactions with plants.Mb can colonize several cereal species, including wheat [29], barley [30], and the model organism Bd [32].Molecular methods for detecting Mb have previously been described by nested PCR [22] and standard PCR [32].In the current study, newly developed primers for qPCR were designed and tested to quantify the amount of Mb DNA in host tissues.The colonization dynamics over time, the distribution of endophytes in different parts of the plant, and the potential of seed transfer, i.e., vertical transfer, were tested in wheat and Bd.Although microscopic methods have been used to determine the presence of endophytes, in many cases, they are combined with molecular or other methods [31,46,47].However, light microscopy could be time-consuming and inaccurate compared to molecular techniques.Identifying individual organisms using microscopy requires considerable experience, as species can only be distinguished based on morphological characters in this way.In addition, individual roots are usually not evenly colonized along their entire length, so only a particular part of the roots needs to be selected for microscopy.If the roots are too thick, focusing the slide in all aspects is challenging.Microscopic detection should be used as a companion tool with a more reliable and accurate molecular method but not as a stand-alone method for diagnosing endophytes.
Mb is a partially saprotrophic organism; in some cases, slightly pathogenic effects on wheat have been observed [30,48].The presence of Mb has been previously described in roots [30], crowns [49], and stems of cereals [50].We used newly developed primers to screen the dynamics of Mb colonization of plants.First, massive colonization of the seed coats occurs, and only then are colonized roots near the crown and parts of the stem base within 1 cm of the crown.Over time Mb spreads to the distal parts of the roots and, in wheat, to the higher parts of the stem.In the lower parts of the roots, the level of endophyte DNA is more or less the same at all sampling periods without significant changes.Mb content in roots part 1 cm bellow crown increases in the last sampling term, e.g., 150 dai in wheat and 120 and 150 dai in Bd.In aboveground parts, the presence of chlamydospores is confirmed microscopically in the outer sheath of the oldest leaf enveloping the stem.No Mb DNA was detected in leaf blades, peduncles, ears, and seeds, nor was the presence of chlamydospores in these plant parts.Seeds from plants heavily colonized by Mb endophyte were harvested and sown, and plants from these seeds were examined for the presence of Mb by both microscopic and qPCR methods.No Mb endophyte was detected in any parts of the plants sown from these seeds, and therefore, it can be concluded that Mb is not transmitted vertically, i.e., by seeds.
Our study is not the first to address the quantification of endophytes in plant tissues using qPCR.For example, Chow et al. (2018) [51] used qPCR to investigate the effect of endophytes (Diaporthe phaseolorum, Trichoderma asperellum, and Penicillium citrinum) on the pathogen Ganoderma boninense that infects oil palm (Elaeis guineensis).The analysis showed that when plants were inoculated simultaneously with endophytes, and the pathogen G. boninense, colonization by the pathogen was suppressed by endophytic fungi, as reflected by the higher DNA content of endophytes compared to that of the pathogen.Latz et al. (2021) [52] investigated interactions between plant genotypes and the biotic and abiotic environment affecting the plant microbiome using the ITS1 metabarcoding method.Root microbial communities were less affected by abiotic factors than the phyllosphere microbiome.The diversity was highest in roots and lower in seeds, while leaves harbored the least diverse microbiome.The complete opposite was found in the work of Tao et al. (2008), who studied the distribution of fungal communities within leaf and root tissues of Bletilla ochracea (Orchidaceae) using random cloning, combined with DGGE (denaturing gradient gel electrophoresis) and phylogenetic analysis [53].The main objective of their study was to compare the diversity of endophytes among roots and leaves of the same plants and to determine whether there is a consistency of communities within different organs of the same host.Fungal communities within leaf and root tissues differed significantly, and diversity within leaves was higher than within roots [53].Bayman et al. (1997) used traditional isolation techniques and found that Xylaria spp.(Xylariales) and Rhizoctonia-like taxa (Basidiomycota) made up the majority of endophytes in epiphytic orchids, and fungal communities within leaves and roots were surprisingly similar [54].
These studies highlight the importance of conducting thorough investigations on endophytes and developing precise and reliable methods for detecting these organisms within the tissues of host plants.The use of molecular techniques in the determination of endophytes has wide applications.Cook et al. (2009) [55] used qPCR to determine the amount of an endophyte involved in producing a toxic alkaloid (swainsonine) in plants of the genus Oxytropis and Astragalus.The endophyte Undifilum oxytropis, involved in synthesizing the toxic alkaloid swainsonine, was quantified in plants of Oxytropis sericea and Astragalus mollissimus species using quantitative PCR.The amount of endophytes varies from one plant part to another and, in some cases, does not correspond to the concentration of swainsonine in the respective parts.The above works deal with microbial diversity inhabiting different parts of plants.Our work focused on one endophyte species and used qPCR to investigate the colonization dynamics and its distribution in the plant.The current study confirmed the endophyte presence in the roots and stem bases of wheat and Bd.Mb is found in roots, generally typical of the fourth class of endophytes to which this endophyte belongs [4].Developing these newly designed primers has significantly broadened our understanding of the dynamics and distribution of endophytes within plants.Moreover, their application extends beyond our study, as they hold great potential for confirming successful colonization in commercial inoculations and facilitating further research in this field.
Fungal endophytes could be transmitted from plant to plant horizontally by soil or vertically by seed.A previous study by Sharon et al. (2023) [2] measured the correlation between fungal endophyte communities in seeds and stems of three cereal species under natural conditions and a controlled environment.Although most dominant fungal endophytic taxa could reach the seed internally, external seed infection (horizontal transfer) was found to be a primary source of specific taxa, including the most abundant species, Alternaria infectoria.Vertical transmission is not probably so frequent and is restricted to particular fungal taxa such as Epichloë [56].Nevertheless, several studies indicate relatively more widespread vertical transfer than previously assumed [57,58].Mb belongs to the category of type 4 endophytes, and like other species in this group, it is recognized for its ability to be horizontally transmitted [4].This has also been confirmed in our study.However, dead plants and debris remain on the ground and can be colonized by Mb.Murray and Gadd (1981) found that colonized seed remnants and roots remain in the ground after the crop is harvested and would provide a source of inoculum for the infection of barley seedlings in the following spring [30].Regarding possible biological control, a potential approach could involve inoculation and colonization of seeds by Mb before sowing.By colonizing the seed coats, these cortical seed parts could serve as a source of inoculum and provide a favorable environment for seedlings' inoculation.However, this hypothetical inoculation method has not yet been developed, and it would be helpful to test it in practice.
There are several reasons why the endophytic fungus Mb and plant Bd create an optimal model for studying plant-fungal interactions.The current study confirmed that the correspondence of Mb and Bd is excellent, and the fungus grows readily in Bd tissues.The inoculation method is simple, and the structures of Mb are readily visible in host tissues using light microscopy.In addition, we have developed and optimized species-specific primers for standard PCR [32] and here also for quantification of Mb by qPCR.The plant colonization dynamics by Mb are thoroughly studied in the current study.Previous studies have shown the beneficial effect of Mb on improving host plant immunity.Optimized methods and available primers from the last research [59,60] allow the analysis of gene expression of specific marker genes in Bd.The advantage of Mb compared to strictly biotrophic symbiotic organisms is the possibility of cultivation on axenic media, and its massive sporulation ability makes its use suitable for largescale production.Our results show that Bd and endophytic fungus Mb can be a model system for the plant-fungi interaction surveys.In general, a more profound knowledge of colonization dynamics and endophyte transmission could be used to improve agricultural management regarding plant growth promotion and biocontrol.

Conclusion
Mb is a suitable model for studying the interactions of endophytic fungi and plants.Primers for qPCR have been developed for the diagnosis and relative quantification of endophyte Mb in plant tissues and tested in wheat and Bd.The method is suitable for confirming the presence of endophytes in plants, studying colonization dynamics and distribution in plants, and other applications.

Table 1 . Fungal species used in the analysis with the MbqITS primers designed in this study. A
positive reaction result is indicated by a Cq lower than 30.The Cq levels in the table are the average of three replicates.